Annotation of the human sequence HS307871 Practical exercise Enrique Blanco - eblanco@imim.es Roderic Guigo - rguigo@imim.es
Abstract: In this exercise, a previously annotated gene will be used to measure the accuracy of different gene finding approaches. GRAIL, GENSCAN, geneid, FGENESH, GenomeScan, GrailEXP and GENEWISE will be used to annotate the sequence. Search by signal, content and homology (protein and cDNA sequences) methods will be employed in order to improve the ab initio results. Weak conservation of Start codons will lead to wrong prediction of initial exons in most cases. Colour legend: Genomic element Operations or links
A. GENE ANNOTATION
Step 1. Accesing EMBL database to retrieve the gene Go to the EMBL database Select Access and SRS Type the accession number for this sequence U30787 Press the Search button Click on the Embl:Entry link Have a look at the different entry fields: find the mRNA and CDS fields. Go to the EMBL Fetch box on the EMBL index page to see the plain text output This is the sequence in FASTA format
B. EXPLORING AB INITIO GENE PREDICTION
Step 2. Running geneid Go to the geneid server Paste the FASTA sequence Choose the geneid output format Run geneid with different parameters: Searching for signals: Select acceptors, donors, start and stop codons. Look for them in the real annotation of the sequence Searching for exons: Select All exons and try to find the real ones Finding genes: You do not need to select any option (default behaviour). Compare the predicted gene with the real gene Figure 1. Signals, exons and genes predicted by geneid in the sequence HS307871
Step 3. Running other genefinders Given that there are several alternative programs to analyze a DNA sequence, we can run a series of applications and observe the common parts of the predictions. GENSCAN: Go to the GENSCAN server Paste the DNA sequence Press the Run Genscan button Compare the annotations and predictions FGENESH: Go to the Softberry homepage In the left frame, select GENE FINDING in Eukaryota Select the program FGENESH Paste the DNA sequence Press the Search button Compare the annotations and predictions GRAIL: Go to the GrailEXP homepage Activate the Perceval Exon Candidates box Paste the DNA sequence Press the Go! button Check the results Compare the annotations and predicted exons NOTE: the first exon is always missed in the predictions and there are some problems to detect the donor site from exon 5. The detection of Start codons is a serious drawback in current gene finding programs (see Figure 2). However, this problem can be overcome by using homology information to complete the gene prediction. Figure 2. EMBL annotation and genes predicted by Grail, GENSCAN, geneid and FGENESH in the sequence HS307871
C. USING EST/cDNA HOMOLOGY INFORMATION
Step 4. Using GrailEXP Go to the GrailExp homepage Activate the Galahad EST/mRNA/cDNA Alignments box Select the GrailEXP database (RefSeq/HTDB/dbEST/EGAD/Riken) Activate exon assembly: Gawain Gene Models Paste the DNA sequence Press the Go! button Check the results: predictions and supporting information Compare annotations, ab initio GRAIL prediction and five predicted alternatively spliced variants Figure 3. Comparison between EMBL annotation and genes predicted ab inition by Grail Vs five alternative predictions supported by ESTs information in the sequence HS307871
Step 5. Using other gene finding programs + alignment of transcripts Using blastn, we can search the database est_human for ESTs supporting future predictions. Filter this output in order to select those non-overlapping ESTs that could form a complete cDNA sequence (see Figure 4). Moreover, ESTs not divided into two or more pieces in the genomic sequence (containing a couple of splice sites) should be rejected. Go to the FGENESH-C server (in Gene finding with similarity menu) Paste the sequence HS307871 Paste the cDNA sequence or EST you have selected Press the search button Note that predicted genes will necessarily be supported by homology information, so it will, most likely, only map to the genomic region overlapping your EST query. Figure 4. Best human ESTs in the alignment mapped on the genomic sequence HS307871
D. Using protein homology information
Step 6. Spliced alignment Spliced alignment is very useful when we have additional information (a putative homologous protein sequence) about the content of the sequence. Gene prediction is then guided by fitting the protein sequence into the best splice sites predicted in the genomic sequence. Open the NCBI blast server Choose the blastx program (genomic query versus protein database) Paste the genomic sequence and press the Blast! and Format! buttons Select second protein (the first one seems to be a truncated isoform). Display the FASTA sequence or click here. Obviously, it is the real protein annotated in the genomic sequence. Open the genewise web server to use this protein to predict the best gene structure Paste both the protein and genomic sequence and run the program Compare the predicted gene (end of the file) and annotations: look for splice sites within introns to check exon boundaries are correct Figure 5. Best HSPs representing proteins homologues similar to the genomic sequence HS307871 obtained using blastx
Step 7. Spliced alignment using homologous proteins From the blastx output, choose several homologous genes and run genewise for each one separately, again. Observe the increase of accuracy when the homologue used is closer to the original human protein: Homo sapiens Ovis aries Mus musculus Rattus norvegicus Danio rerio Drosophila melanogaster Drosophila virilis Saccharomyces cerevisiae Schizosaccharomyces pombe Figure 6. Graphical comparison of the real gene annotation and different genewise predictions using different homologous proteins for the gene uroporphyrinogen decarboxylase (URO-D)
Step 8. Spliced alignment using an homologous cDNA As with protein homology information, we can also use an homologous cDNA to do a spliced alignment. Go to the Spidey web server. Select the homologous mouse cDNA. Paste both the human genomic and the mouse cDNA sequence in the appropriate fields. Presss the Align button Check the results
Step 9. Using protein homology information: GenomeScan Protein homology information can also be used to enhance ab initio predicted exons supported by blastx HSPs as in the case of GenomeScan and geneid, thereby improving the final prediction GenomeScan: Go to the GenomeScan web server Retrieve the protein from the previous blast search Paste both genomic and protein sequences Press the button GenomeScan Check the results. It seems that the first exon has not been detected even using homology information. This is due to the fact that blast programs have a minimal word length. Figure 6. GenomeScan output: first exon is not correctly predicted probably due to blast word length restrictions
E. USING A GENOME ANNOTATION BROWSER
Step 9. Golden path archive: Open the UCSC Genome Bioinformatics Site Select the blat link to locate the genomic coordinates of our sequence Paste the DNA sequence in FASTA format (HS307871) Submit the query Click on the first hit: (browser link) Compare the graphical annotation with the EMBL entry of the gene Analyze these different sets of output options: Genes and Gene Prediction Tracks, mRNA and EST Tracks Figure 7. (a) UCSC genome browser representation of the region containing the gene uroporphyrinogen decarboxylase (URO-D) (b) UCSC genome browser representation of the context (100Kbps) region around the gene uroporphyrinogen decarboxylase (URO-D).
F. RESULTS
Here you can find the solutions for every exercise: EMBL annotation EMBL annotation (plain text) FASTA sequence geneid results: signals geneid results: exons geneid results: genes GENSCAN results FGENESH results GRAIL results GrailEXP results Blastn + human ESTs results Blastx + protein results Genewise (human protein) Genewise (ovis protein) Genewise (mouse protein) Genewise (rat protein) Genewise (Danio rerio protein) Genewise (Drosophila melanogaster protein) Genewise (Drosophila virilis protein) Genewise (yeast protein) Genewise (fission yeast protein) GenomeScan results
G. BIBLIOGRAPHY
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